The continued increase in marijuana abuse has created an even greater demand for sensitive, rapid and reliable methods for confirming the presence of this drug in biological samples. Confirmation of the presence of the drug is important in initially detecting marijuana users as well as assisting drug counseling programs in monitoring compliance with rehabilitation programs.
Marijuana abuse is detected by identifying the presence of metabolites of the major psychoactive constituent of marijuana, delta-9-tetrahydrocannabinol, in biological fluids. Urine, because of its less invasive and more convenient sampling process, is the biological fluid most often analyzed for marijuana abuse. The major metabolite of delta-9-tetrahydrocannabinol found in urine is 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) existing in both a free acid and a conjugated (glucuronide) form. Many analytical procedures using gas chromatography, high performance liquid chromatography, thin layer chromatography, gas chromatography-mass spectrometry, radioimmunoassay and enzyme multiplied immunoassay have been developed for determining the presence of THC-COOH in urine. However, the sensitivity and, more importantly, the reliability of these methods are hindered by inadequacies of the techniques used in preparing urine samples for analysis. Current methods of sample preparation, using techniques such as thin layer chromatography, liquid-liquid extraction and other solid phase extraction techniques commonly suffer from low drug recovery, incomplete removal of interfering urine components, and/or long preparation times.
Measurement of THC-COOH in urine is considerably complicated by the complexity of the urine matrix. The large number of organic acids present in urine make extraction of THC-COOH quite difficult. Many of these organic acids exhibit chromatographic properties similar to THC-COOH and therefore may interfere with the measurement of the drug metabolite. Therefore, selective extraction of THC-COOH from the urine sample matrix is essential to providing the sensitivity and reliability required for confident drug monitoring.
Typical urine screens for THC-COOH and many other abused-drugs are performed using thin layer chromatography or the Enzyme Multiplied Immunoassay Technique (EMIT) of Syva Company. Positive results are then confirmed using a second more specific test. Confirmation is usually obtained using gas chromatography-mass spectrometry techniques, however other chromatographic techniques may be employed. The use of these techniques has created a need for a more efficient sample cleanup technique. A bonded phase chromatographic packing that allows selective washing of impurities without removing the THC-COOH and/or permits selective elution of the THC-COOH without removing impurities would be highly desirable. A clean, concentrated extract with good metabolite recovery will allow for much more sensitive confirmation analyses. A clean extract will also aide in maintaining the integrity of the instrumentation used in confirmation.
Several existing patents, such as U.S. Pat. Nos. 4,640,909; 4,650,784; 4,680,120 and 4,680,121, and journal publications, such as M. ElSohly et al, Analysis of the Major Metabolite of delta-9-tetrahydrocannabinol in Urine, Journal of Analytical Toxicology, vol. 7, November/December 1983, pp. 262-264; J. Vinson et al, A Semi-Automated Extraction and Spotting System for Drug Analysis by TLC, Journal of Analytical Toxicology, vol. 9, January/Febuary 1985, pp. 6-7 H. McCurdy et al, Evaluation of the Ion Trap Detector for the Detection of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid in Urine After Extraction by Bonded-Phase Adsorption, Journal of Analytical Toxicology, vol. 10, September/October 1986, pp. 175-177, report the use of solid phase extraction in the analysis of THC-COOH in urine. All of this prior art uses bonded phases and extraction procedures significantly different from those represented in this invention.
The four United States patents, held by J. T. Baker, disclose the use of silica based bonded phases with ether, ester, thiol ether and alkyl function groups for extracting THC-COOH from urine using a reverse phase or hydrogen bonding mechanism. The processes for the extraction of the drug metabolite also use different solutions and solvents for conditioning and washing the bonded phase as well as eluting the drug metabolite from the bonded phase than those disclosed in this invention.
The work of ElSohly et al and McCurdy et al discuss the use a bonded phase, produced by Analytichem International, for the extraction of THC-COOH from urine. The chemistry of the bonded phase is not disclosed in this work but once again different solutions and solvents are used in the conditioning, washing and elution of the bonded phase than those used in this invention. The work of Vinson et al discusses the use of an ion exchange resin in the extraction of THC-COOH. Once again the extraction process differs significantly from the process disclosed in this invention.
The invention of this document uses a unique bonded phase which contains a sulfonylazide grafted to silica through a reversed phase side chain which isolates THC-COOH from urine. Absolute recovery of the THC-COOH is also significantly improved when using the invention as compared to the recoveries obtained from the previously published methods.